A new method, based on the use of cationic beads has been developed to isolate cell membranes. During the next project period this method will be perfected and used as the basis for bulk freeze-fracturing splitting membranes and purifying milligram quantities of extracellular and protoplasmic half-membranes. As part of this effort, transbilayer exchange and the maintenance of membrane asymmetry will be measured in large lipid bilayer vesicles, and in lipid bilayers and erythrocyte membranes on cationic beads. Membrane splitting will be measured using fluorescence resonance energy transfer. The techniques to be developed will provide new methods of studying the molecular basis of membrane organization and function.